RESUMO
BACKGROUND/AIM: Microcirculation in the dermis of the skin is important for nutrient delivery to this tissue. In this study, the effects of a micronutrient concentrate (Juice Plus+®; 'active group'), composed primarily of fruit and vegetable juice powder, on skin microcirculation and structure were compared to placebo. STUDY DESIGN/METHODS: This 12-week study had a monocentric, double-blind placebo and randomized controlled design with two treatment groups consisting of 26 healthy middle-aged women each. The 'oxygen to see' device was used to evaluate microcirculation. Skin density and thickness were measured using ultrasound. Measurements for skin hydration (Corneometer®), transepidermal water loss and serum analysis for carotenoids and α-tocopherol were also performed. RESULTS: By 12 weeks, microcirculation of the superficial plexus increased by 39%. Furthermore, skin hydration increased by 9% while skin thickness increased by 6% and skin density by 16% in the active group. In the placebo group, microcirculation decreased, and a slight increase in skin density was observed. CONCLUSION: Ingestion of a fruit- and vegetable-based concentrate increases microcirculation of the skin at 12 weeks of intervention and positively affects skin hydration, density and thickness.
Assuntos
Suplementos Nutricionais , Frutas , Microcirculação/efeitos dos fármacos , Pele/efeitos dos fármacos , Verduras , Adulto , Idoso , Carotenoides/sangue , Carotenoides/farmacocinética , Carotenoides/farmacologia , Método Duplo-Cego , Feminino , Humanos , Micronutrientes/sangue , Micronutrientes/farmacocinética , Micronutrientes/farmacologia , Pessoa de Meia-Idade , Pele/irrigação sanguínea , Pele/diagnóstico por imagem , Ultrassonografia , alfa-Tocoferol/sangue , alfa-Tocoferol/farmacocinética , alfa-Tocoferol/farmacologiaRESUMO
OBJECTIVES: Oxidative stress is suggested to play an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH). The present study was aimed to compare plasma levels of antioxidants in patients suffering from NASH and healthy controls. METHODS: Plasma levels of the antioxidants α-tocopherol, γ-tocopherol, lutein, zeaxanthin, ß-cryptoxanthin, lycopene, α-carotene ß-carotene were determined in 57 patients with biopsy-proven NASH and 40 healthy controls. RESULTS: Levels of α-tocopherol (22.4 vs. 26.8 nmol/ ml; p<0.01), lutein (0.19 vs. 0.33 nmol/ml; p<0.0001), zeaxanthin (0.04 vs. 0.08 nmol/ml; p<0.0001), lyco?pene (0.15 vs. 0.42 nmol/ml; p<0.0001), α-carotene (0.03 vs. 0.06 nmol/ml; p<0.005) and ß-carotene (0.25 vs. 0.39 nmol/ml; p<0.01) were significantly decreased in NASH patients compared to controls. Age, aminotransferase status (ALT, AST) and BMI were not correlated with the levels of tocopherols or caro?tenoids. CONCLUSIONS: Given the decreased levels supplementation of lipophilic antioxidants might be a rational treatment option for patients with NASH.
Assuntos
Antioxidantes/metabolismo , Carotenoides/sangue , Fígado Gorduroso/sangue , Vitamina E/sangue , Fígado Gorduroso/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologiaRESUMO
Modern metabolic research in hepatology employs non-invasive techniques of the whole organism, and it includes studying the intact organ. Following recent decades of efforts in culturing isolated cells and studying their properties separately, it has become clear that the spatiotemporal organisation of different cell types in a tissue requires studies using models of the intact organ or tissue. This applies particularly to the liver as the major organ of metabolic transformation and activity. The present brief article attempts to illustrate the advantages and limitations of such approaches, focusing on selected aspects of ammonia/urea and glutamine metabolism as an example.
Assuntos
Gastroenterologia/tendências , Fígado/metabolismo , Modelos Animais , Técnicas de Cultura de Órgãos/métodos , Técnicas de Cultura de Órgãos/tendências , Perfusão/métodos , Perfusão/tendências , Animais , HumanosRESUMO
Ultraviolet A (UVA) irradiation is effectively used to treat patients with atopic dermatitis and other T cell mediated, inflammatory skin diseases. In the present study, successful phototherapy of atopic dermatitis was found to result from UVA radiation-induced apoptosis in skin-infiltrating T helper cells, leading to T cell depletion from eczematous skin. In vitro, UVA radiation-induced human T helper cell apoptosis was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation. These studies demonstrate that singlet oxygen is a potent trigger for the induction of human T cell apoptosis. They also identify singlet oxygen generation as a fundamental mechanism of action operative in phototherapy.
Assuntos
Apoptose/imunologia , Apoptose/efeitos da radiação , Oxigênio/farmacologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/efeitos da radiação , Terapia Ultravioleta , Anticorpos Bloqueadores/farmacologia , Apoptose/efeitos dos fármacos , Dermatite Atópica/imunologia , Dermatite Atópica/radioterapia , Deutério/farmacologia , Proteína Ligante Fas , Humanos , Ligantes , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/efeitos da radiação , Naftóis/farmacologia , Oxigênio Singlete , Azida Sódica/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(-4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes.
Assuntos
Exocitose , Aparelho Lacrimal/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Peroxidases/metabolismo , Animais , Atropina/farmacologia , Carbacol/farmacologia , Dinitrofenóis/farmacologia , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/ultraestrutura , Masculino , Consumo de Oxigênio , Ratos , Receptores Colinérgicos , TemperaturaRESUMO
Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.
Assuntos
Antagonistas de Receptores de Andrógenos , Óxido Nítrico/fisiologia , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Imuno-Histoquímica , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Neoplasias da Próstata/enzimologia , Receptores Androgênicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We designed a genetically encoded ratiometric fluorescent probe, SypHer3s, with enhanced brightness and optimized pKa, which responds to pH changes in different cellular compartments. SypHer3s was successfully utilized for imaging the pH dynamics in mitochondria of living neurons and in quantitative pH measurement in zebrafish embryos.
RESUMO
Expression of the oncogenes, epidermal growth factor (EGF) receptor, HER2/neu, c-myc, and c-fos, in renal cell carcinoma and corresponding nonneoplastic kidney tissue of 30 patients has been analyzed by Northern blot analysis. In renal cell carcinoma an inverse relationship of EGF receptor and HER2/neu gene expression was detected, with high expression of the EGF receptor gene in 22 of 30 (73%) cases and low expression of the HER2/neu gene in 28 of 30 (93%) cases. Furthermore, altered expression of the oncogenes c-myc and c-fos was detected in renal cell carcinoma, which appears to be related to the tumor grade of malignancy. Additional Southern blot analysis of six renal cell carcinomas gave no indication of chromosomal rearrangement events or gene amplification.
Assuntos
Carcinoma de Células Renais/genética , Receptores ErbB/genética , Expressão Gênica , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Biomarcadores Tumorais/análise , Northern Blotting , Southern Blotting , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Hibridização de Ácido Nucleico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-myc , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Receptor ErbB-2RESUMO
5-Aminolevulinate (ALA) photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. ALA is applied as a precursor of porphyrins serving as endogenous photosensitizers. Irradiation of HaCaT cells preincubated with 1 mM ALA for 24 h with red light of 570-750 nm at a dose of 4.5 J/cm2 leads to a 6-fold elevation of cellular c-Jun N-terminal kinase activity; phosphorylation of p38 mitogen-activated protein kinase (MAPK) is enhanced to a similar extent. In contrast, neither activation nor increased phosphorylation of the extracellular stimulus-regulated kinase MAPKs is detected. p38 is also phosphorylated by ALA-PDT in the human melanoma cell lines Bro and SkMel-23, applying doses that lead to 80-95% cell death after 24 h. Hence, the effects of ALA-PDT on MAPKs are similar to stresses like UV irradiation or exposure to hydrogen peroxide with respect to activation of JNK and p38 MAPKs. They are different, however, in that extracellular stimulus-regulated kinase activity is not raised by ALA-PDT. Of the 830 pmol porphyrins/mg protein that were present at 24 h in HaCaT cells, 99 pmol/mg were intracellular. When extracellular porphyrins had been removed by washing, p38 responses were retained. Thus, intracellular porphyrins synthesized from ALA are sufficient to elicit activation of p38 on photosensitization.
Assuntos
Ácido Aminolevulínico/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Fotoquimioterapia , Pele/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Queratinócitos , Melanoma , Fosforilação , Pele/enzimologia , Pele/efeitos da radiação , Neoplasias Cutâneas , Células Tumorais Cultivadas , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Human renal cell carcinoma is characterized by the loss of differentiation markers such as glutathione S-transferase alpha (GST-alpha). In this paper we show that the promoter of a GST-alpha gene contains a functional binding site for the cell-specific transcription factor LFB1 (HNF1). To investigate the potential role of LFB1 in the down-regulation of GST-alpha expression, we have compared the amount and the binding activity of the LFB1 protein between normal kidney and tumor tissue. By Western analysis and gel retardation assay using a monoclonal antibody specific for LFB1 we show that in 11 of 14 carcinomas the amount of LFB1 is clearly reduced compared to the corresponding normal tissue and that in all 14 renal carcinomas LFB1 binding activity is diminished. As in the same samples the abundance of GST-alpha mRNA is lower than in the normal tissue, we postulate that the loss of LFB1 binding activity might be responsible for the decreased expression of the GST-alpha gene in renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Proteínas Nucleares , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Anticorpos Monoclonais , Especificidade de Anticorpos , Sequência de Bases , Sítios de Ligação , Carcinoma de Células Renais/enzimologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Humanos , Rim/metabolismo , Rim/fisiologia , Neoplasias Renais/enzimologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/genéticaRESUMO
1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.
Assuntos
Glutamato-Amônia Ligase/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Fígado/enzimologia , Cloreto de Amônio/farmacologia , Animais , Glucagon/farmacologia , Cinética , Fígado/efeitos dos fármacos , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
A method for the synthesis of the glutathione conjugate S-(4-azidophenacyl)[35S]glutathione is described. The compound was used for photoaffinity labeling of proteins present in canalicular membrane vesicles (CMV), sinusoidal membrane vesicles (SMV), mitochondria and microsomes from rat liver. Most of the radioactivity introduced by photoaffinity labeling of CMV appeared in the 25-29 kDa range. Further labeled proteins were observed in bands at 37, 105 and about 120 kDa. 79% of the 25-29 kDa associated radioactivity was recovered in the supernatant after extensive revesiculation (washing) of the vesicles, together with the 37 kDa protein. CMV and SMV contained glutathione S-transferase (GST) activity which in CMV was decreased by 75% by washing. Photolabeling of a mixture of purified basic GST subunits from rat liver resulted in a band pattern at 25-29 kDa similar to that in the membrane preparations. Isoelectric focusing of the CMV indicated the presence of basic soluble GST subunits. S-Hexylglutathione-Sepharose affinity chromatography showed reversible binding of photolabeled proteins at 25-29 kDa. Difference photoaffinity labeling with GSSG, S-hexylglutathione, taurocholate and phenylmethylsulfonyl fluoride decreased the radioactivity bound by GST, but not that introduced into the 105 kDa protein band present in CMV. It is concluded that membrane-associated basic GST isoenzymes are present in standard membrane vesicle preparations. In the cell, the function may be transport of GST-bound compounds across the membrane and protection of the membranes against electrophiles.
Assuntos
Marcadores de Afinidade/síntese química , Glutationa/análogos & derivados , Fígado/metabolismo , Animais , Azidas , Fracionamento Celular , Membrana Celular , Cromatografia de Afinidade , Glutationa/síntese química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Técnicas In Vitro , Proteínas de Membrana/metabolismo , Peso Molecular , Fotoquímica , RatosRESUMO
Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside-out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio-3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high-affinity transporter for DNP-SG and GSSG in erythrocytes is suggested.
Assuntos
Membrana Eritrocítica/metabolismo , Glutationa/análogos & derivados , Transporte Biológico , Cloromercurobenzoatos/farmacologia , Ácido Ditionitrobenzoico/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Etilmaleimida/farmacologia , Fluoretos/farmacologia , Glutationa/metabolismo , Dissulfeto de Glutationa , Temperatura Alta , Humanos , Cinética , Especificidade por Substrato , Vanadatos/farmacologiaRESUMO
To study the formation of DNA methylation patterns, plasmids containing promoters of different strengths in front of the bacterial chloramphenicol acetyltransferase reporter gene were transfected into F9 mouse embryonal carcinoma cells. Methylation of the integrated plasmids as well as copy numbers and activities of the reporter gene were determined for individual cell clones. The methylation pattern of the integrated plasmids was found to be determined by properties of the DNA sequence itself. In contrast, the specific methylation patterns were invariant with respect to integration site, copy number and arrangement of the integrates; methylation did also not correlate with transcriptional activity of the different promoters. Certain promoter regions may therefore contain signals recognized by the de novo methylation activity in embryonal carcinoma cells.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Células-Tronco Neoplásicas/enzimologia , Plasmídeos , Animais , DNA/isolamento & purificação , Células-Tronco de Carcinoma Embrionário , Metilação , Camundongos , Regiões Promotoras Genéticas , Transcrição Gênica , TransfecçãoRESUMO
The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.
Assuntos
Peróxidos Lipídicos/metabolismo , Microssomos/efeitos dos fármacos , Ácido Tióctico/análogos & derivados , Vitamina E/farmacologia , Animais , Medições Luminescentes , Microssomos/metabolismo , Ratos , Tiobarbitúricos , Ácido Tióctico/farmacologia , Fatores de TempoRESUMO
ATP-stimulated uptake of S-(2,4-dinitrophenyl)glutathione with a high activity of 0.35 nmol/min per mg protein is found in a rat liver plasma membrane vesicle preparation enriched in sinusoidal marker enzymes. Transport takes place into an osmotically active space. Vanadate and S-(azidophenacyl)glutathione inhibit transport, whereas Ca2+, EGTA and ouabain are without effect.
Assuntos
Trifosfato de Adenosina/farmacologia , Membrana Celular/metabolismo , Glutationa/análogos & derivados , Fígado/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Glutationa/metabolismo , Cinética , Concentração Osmolar , RatosRESUMO
The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Músculos/metabolismo , Fosfocreatina/metabolismo , Animais , Ciclo do Ácido Cítrico , Creatina/metabolismo , Creatina Quinase/metabolismo , Estimulação Elétrica , Glicólise , Concentração de Íons de Hidrogênio , Masculino , Contração Muscular , Músculos/fisiologia , Esforço Físico , Ratos , Ratos Endogâmicos , Frações Subcelulares/metabolismoRESUMO
Rat liver plasma membranes exhibit membrane-bound glutathione S-transferase activity. The specific activity in isolated canalicular membranes was 83 +/- 8 mU/mg protein and 50 +/- 3 mU/mg protein in the sinusoidal membranes. Whereas microsomal and outer mitochondrial glutathione S-transferases were stimulated seven and four-fold with N-ethylmaleimide, respectively, the plasma membrane activity was activated two-fold. Western blot analysis, using an antibody against the microsomal glutathione S-transferase, shows the presence of a 17 kDa protein in canalicular and sinusoidal membrane fractions. The antibody reaction was about three-fold higher in the canalicular compared to the sinusoidal membrane fraction. These data support the conclusion that the plasma membrane glutathione S-transferase is closely related to the microsomal and outer mitochondrial membrane enzyme.
Assuntos
Membrana Celular/enzimologia , Etilmaleimida/farmacologia , Glutationa Transferase/metabolismo , Fígado/enzimologia , Animais , Western Blotting , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Masculino , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos Wistar , Reagentes de Sulfidrila , Fatores de TempoRESUMO
The biologically occurring thiols, glutathione, cysteamine and cysteine, significantly enhance the single-strand breaks in plasmid pBR322 DNA induced by singlet molecular oxygen (1O2) generated by the thermodissociation of the endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. The enhancing effect was also observed with chemically related sulfhydryl compounds but not by disulfides. In contrast, dihydrolipoate and its disulfide lipoate protected the plasmid DNA. Metal chelators as well as superoxide dismutase or catalase had no effect, whereas mannitol or sodium azide, decreased the thiol-1O2-induced strand breaks. It is concluded that the observed effects are mediated by reactive oxidation products arising from the 1O2-oxidation of thiols.
Assuntos
Dano ao DNA , Oxigênio , Plasmídeos , Compostos de Sulfidrila/farmacologia , Quelantes , Cisteamina/farmacologia , Cisteína/farmacologia , Sinergismo Farmacológico , Eletroforese em Gel de Ágar , Radicais Livres , Glutationa/farmacologiaRESUMO
Damage of plasmid and bacteriophage DNA inflicted by singlet molecular oxygen (1O2) includes loss of the biological activity measured as transforming capacity in E. coli and single-strand break formation. Three different sources of 1O2 were employed: (i) photosensitization with Rose bengal immobilized on a glass plate physically separated from the solution; (ii) thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO2); and (iii) microwave discharge. Loss of transforming activity was documented after exposing bacteriophage M13 DNA to 1O2 generated by photosensitization employing immobilized Rose bengal, and with bacteriophage luminal diameter X174 DNA, using the thermodissociable endoperoxide (NDPO2) as a source of 1O2. These findings are in agreement with experiments in which plasmid DNA pBR322 was exposed to a gas stream of 1O2 generated by microwave discharge. The effects of 1O2 quenchers and of 2H2O indicate 1O2 to be the species responsible. Strand-break formation in pBR322 and luminal diameter X174, measured as an increase of the open circular form at the expense of the closed circular supercoiled form, was observed without alkaline treatment after exposing the DNA to 1O2, using either agarose gel electrophoresis or sucrose gradient separation. The effect of quenchers and 2H2O indicate the involvement of 1O2 in DNA damage. We conclude that singlet oxygen can cause loss of biological activity and DNA strand breakage.